Exosomes sourced from granulocytic myeloid-derived suppressor cells and application thereof

ABSTRACT

Provided are exosomes sourced from a granulocyte myeloid-derived suppressor cell and an application thereof. The exosomes are named as G-MDSC exo. Also provided is a use of the exosomes in preparing a drug used for suppressing autoimmune diseases. The G-MDSC exo can effectively suppress proliferation of CD4+T cells in vitro, promote induced proliferation of T regulatory (Treg) cells, alleviate foot swelling of model mice having delayed-type hypersensitivity, and suppress attacks of inflammatory bowel disease (IBD) and collagen-induced arthritis (CIA) of the mice.

FIELD OF INVENTION

This invention relates to granulocytic-like myeloid-derived suppressorcells derived exosomes (G-MDSC exo) and application thereof, whichbelongs to the fields of cell biology, molecular biology and clinicalapplication. Specifically, the present invention relates to thepreparation of G-MDSC exo from spleens of tumor-bearing mouse, theresearch of biological function and its application in treatment ofmurine autoimmune diseases.

BACKGROUND OF THE INVENTION

Myeloid-derived suppressor cells (MDSCs) are myeloid-derivedheterogeneous cells that proliferate greatly under pathological cases oftumor, inflammation and pathogen infection (Gabrilovich D I, Nagaraj S.Myeloid-derived suppressor cells as regulators of the immune system. NatRev Immunol. 2009; 9(3):162-74.). MDSCs of mouse are Gr-1 (consisting ofLy6G and Ly6C markers) and CD11b double-positive cells. Two subtypes ofMDSCs can be divided into by cell morphology and the expression level ofLy6G and Ly6C: CD11b⁺Ly6G⁺Ly6C^(low) granulocytic like MDSCs (G-MDSCs),and CD11b⁺Ly6G⁻Ly6C^(hi) monocyte like MDSCs (M-MDSCs). Both subtypesexpand greatly under conditions such as tumor and infection, and theexpansion of G-MDSCs is significantly more than that of M-MDSCs. G-MDSCsinhibit T cell-mediated adaptive immune responses and natural anti-tumorimmune responses mediated by natural killer cells (NK) and macrophagethrough expressing arginase 1 (Arg-1) and reactive oxygen species (ROS).Studies have shown that MDSCs can induce regulatory T cells (Tregs)differentiation through interferon γ (IFN-γ) and interleukin 10 (IL-10)dependent-pathway thereby down-regulating function of effector T cells.In recent years, studies show that MDSCs have great potential in thetreatment of autoimmune diseases. Therefore, the therapeutic value ofMDSCs is re-evaluated. MDSCs have been used to treat mousecollagen-induced arthritis (CIA) and showed some efficacy. However, thelack of sources, inconvenience of storage and complex composition limitits application.

Autoimmune diseases are caused by immune system responding to their ownconstituents, which have a high morbidity in the population.Inflammatory bowel disease (IBD) is a non-specific, inflammatoryintestinal disease which is mainly caused by the abnormal immuneresponse to the normal flora which is at tolerable state. IBD includesulcerative colitis (UC) and Crohn's disease (CD). The main clinicalsymptoms are abdominal pain, diarrhea, and bloody mucopurulent stools;long term illness may lead to the development of cancer. Rheumatoidarthritis (RA) is a common clinical systemic autoimmune disease, themain clinical symptoms include symmetry small joint lesion,characterized by persistent inflammation in multiple joints. Largeamount of studies have shown that proinflammatory Th1 and Th17 cells andtheir derived inflammatory factors play important roles in thedevelopment of autoimmune diseases. Tregs play an essential role inmaintaining the intestinal immune balance. The imbalance of Tregsfunction is one of the causes of autoimmune diseases. Studies confirmedthat the number of Tregs in patients with autoimmune diseases is lowerthan healthy persons. In recent years, the incidence of autoimmunediseases is on the rise in China. However, autoimmune diseases patientsmainly receive symptomatic treatment in clinic, but there is noeffective treatment measures, thus exploring an effective and curativetreatment method is urgently desirable.

Exosomes are membranous vesicles released into extracellular environmentafter fusion of intracellular multivesicular complete with the plasmamembrane. It has been demonstrated that almost all living cells cansecrete exosomes, and exosomes widely exist in various body fluids(Beninson L A, M. Fleshner. Exosomes: an emerging factor instress-induced immunomodulation[J]. Semin Immunol, 2014. 26(5):394-401). Exosomes carry protein, mRNA and miRNA and other ingredientsderived from the derived cells, and protect them from degradation by theexternal environment, and are beneficial to their biological function ofactive ingredients. Exosomes can be internalized by receptor cellsthrough giant pinocytosis, lipid rafts and receptor-mediated endocytosisetc. and the internalized exosomes can regulate and control multiplebiological function of receptor cells and play an important role inintercellular communication. The roles of exosomes in the process ofimmune response, apoptosis, angiogenesis, inflammation reaction, andtumor occurrence and development have been reported. Studies showed thatexosomes exhibited more advantages than derived cells when they wereused in disease intervention. It had been reported that dendriticcell-derived exosomes were used to treat cancer and showed good efficacy(Pitt J M, Charrier M, Viaud S, et al. Dendritic cell-derived exosomesas immunotherapies in the fight against cancer [J]. J Immunol, 2014;193(3):1006-11.). However, studies on function of G-MDSC exo and theapplication thereof in the treatment of autoimmune diseases have notbeen reported.

We found that G-MDSC exo has a diameters of 40-100 nm, carriesbiologically active ingredients and the biological characters wereclearer than other G-MDSC secreted vesicles. G-MDSC exo could suppressCD4⁺ T cell proliferation, relieve the foot pad swelling degree ofDelayed Type Hypersensitivity (DTH) mouse, and promote the expansion ofCD4⁺ T cell to Tregs induced by TGF-β. Application of G-MDSC exo in vivocould attenuate the severity of DTH response, IBD and CIA, and furtherdemonstrating that this function depends on Arg-1 carried by G-MDSC exoat certain degree.

In recent years, researches on exosomes in disease treatment mainlyconcentrated on dendritic cells and stem cells derived exosomes. In theaspect of disease treatment, exosomes exhibit more advantages thanderived cells. Firstly, storage and transport of exosomes are easier,has no cytotoxicity, and they can be used with less biosafety problems.Secondly, the complex molecules on the surface of exosomes offerpotential homing mechanisms for specific target tissues andmicroenvironments (Xinrui Tian, Wenqing Tian, Bo Niu, et al. Thepreparation of neural stem cells derived exosomes and applicationthereof in the nervous system diseases. China, CN103740645A[P].2014-04-23). Lastly, exosomes protect the therapeutic proteins andnucleic acids, thereby reducing degradation of the therapeutic proteinsand nucleic acids (Marcus M E, Leonard J N. FedExosomes: EngineeringTherapeutic Biological Nanoparticles that Truly Deliver W.Pharmaceuticals (Basel), 2013, 6(5):659-680.). The application of G-MDSCexo in the treatment of disease has not been reported. Our experimentalresults showed that G-MDSC exo could maintain the biological activityfor a long time at low temperatures. G-MDSC exo not only could suppressCD4⁺ T cell proliferation similar to G-MDSC but also could promote theexpansion of CD4⁺ T cell to Tregs cell induced by TGF-β. G-MDSC exo is anatural immunomodulator, is essential in the treatment of autoimmunediseases and has good application value in clinic.

Based on structural and functional properties of G-MDSC exo, theapplication of G-MDSC exo in the treatment of autoimmune diseases willachieve the purpose of “low-input, high output”. It has a good prospectof clinical application, is expected to play a positive role inmaintaining the body's immune balance. It will provide a new therapeuticapproach for autoimmune disease.

SUMMARY OF THE INVENTION

This invention is to overcome the deficiencies in the prior art, andprovides G-MDSCs derived exosomes (G-MDSC exo). This exosomes havenonspecific components of G-MDSCs and specific active components such asprotein and nucleic acid in G-MDSCs, is a composite informosomes ofsubcellular structure. G-MDSC exo not only possess immunosuppressiveproperties of G-MDSCs and but also contains its own surface moleculeCD63. We have observed its role in the therapy of autoimmune diseases toseek method to treat disease fundamentally.

G-MDSC exo of the present invention are membranous vesicles composed ofderived cell membrane lipid components, which has protection effects ontheir carried bioactive ingredients. G-MDSC exo can playimmunosuppressive roles similar to G-MDSCs, and the exosomes have manyadvantages that G-MDSCs do not have, they are composite informosomes ofsubcellular structure for intercellular information transmission.

The invention discloses a simple method to prepare G-MDSC exo derivedfrom tumors or autoimmune disease individuals. The whole process isshort time-consuming, high yield. G-MDSC exo is easy for storage and hasstable biological activity. This method provides the basis for massiveproduction of G-MDSC exo for therapeutic purposes. Briefly, weconstructed mouse tumor or autoimmune disease models (e.g. CIA),isolated G-MDSCs from spleens, collected culture supernatants, andfinally extracted G-MDSC exo with combination of ultracentrifugationcentrifugation and microporous membrane filter.

The detailed procedures are as follows:

The collected G-MDSCs culture suspension was centrifuged at 4° C. andthen collected the supernate, repeating 3 times. The supernatantfraction was transferred to MWCO 100 kDa ultrafiltration centrifugetube, and then collect the concentrated solution in the tube aftercentrifugation. The concentrated solution was mixed with ExoQuick-TC™Exosome reagent (v/v=5:1), vibrated and stewed at 4° C., the supernatantwas discarded after centrifugation at 4° C., collecting the sediment toobtain exosomes.

G-MDSC exo in the present invention suppress T cell proliferation andpromote Tregs polarization. Application of G-MDSC exo in vivo may playstrong immunosuppressive role and is essential in the therapy ofautoimmune diseases.

G-MDSC exo in the present invention could effectively alleviate clinicaldevelopment of DTH, IBD and CIA in mouse, reducing pathological damage.The present invention provides a novel pathway to treat autoimmunediseases by restoring the immune balance.

This invention also confirmed that G-MDSC exo exhibited theimmunosuppressive effects by inhibiting CD4⁺ T cell proliferation andpromoting Treg cell expansion. G-MDSC exo plays a protective effect onautoimmune diseases and provides a new approach for the treatment ofautoimmune diseases.

It is an advantage that the present invention prepares G-MDSC exo fromtumors or autoimmune disease individuals, and it lays the foundation forits application and associated biological function investigation.

It is another advantage, compared with the conventional differentialcentrifugation, the process of preparing G-MDSC exo in the presentinvention is short time-consuming, simple, convenient for large scaleextraction, and the structure of G-MDSC exo is complete.

In is another advantage that the active ingredients contained in G-MDSCexo are stable and easy to preserve, when stored at −80° C. it canpreserve for more than one year. At room temperature, the structure andactivity of G-MDSC exo are stable, and they are easy to transportwithout cryopreservation and resuscitation. All these advantages providea technical basis for the clinical application of G-MDSC exo.

It is still another advantage that G-MDSC exo is a nano-grade compositeinformosomes of subcellular structure without cytotoxicity, has a highclinical safety.

It is yet another advantage that G-MDSC exo has a therapeutic effect onautoimmune diseases, its preparation and application provides a new wayfor the treatment of autoimmune diseases.

It is a further advantage that G-MDSC exo inhibits CD4⁺ T cellproliferation and promotes Treg expansion, it provides a new concept forthe treatment of autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Purity of G-MDSCs sorted by immune magnetic beads and detectedby fluorescence activated cell sorter (FACS) analysis.

FIG. 2: Identification of G-MDSC exo. A: Morphology of G-MDSC exo; B:Particle-size and frequency distribution of G-MDSC exo; C: Expression ofG-MDSC exo detected by western blot.

FIG. 3: G-MDSC exo suppresses CD4⁺ T cell proliferation. A: Effect ofG-MDSC exo on suppressing CD4⁺ T cell proliferation measured by[³H]-thymidine incorporation method through in vitro observation; B:Effects of G-MDSC exo on suppressing the DTH model through in vivoobservation.

FIG. 4: G-MDSC exo promotes the expansion of CD4⁺ T cells to Tregs cellsinduced by TGF-β in a dose-dependent manner. Figure A shows G-MDSC exopromotes the expansion of CD4⁺ T cells to Tregs cells induced by TGF-βof different concentration. Figure B shows G-MDSC exo promotes theexpansion of CD4⁺ T cells to Tregs cells induced by TGF-β in adose-dependent manner.

FIG. 5: The protective effect of G-MDSC exo to IBD mouse. A: Effect ofG-MDSC exo on the disease progression of IBD mouse; B: Effect of G-MDSCexo on the colon tissues of IBD mouse; C: Effect of G-MDSC exo oncolonic inflammation of IBD mouse.

FIG. 6: The protective effect of G-MDSC exo on CIA mouse. A:Experimental flow diagram of CIA mouse intervened with G-MDSC exo. B:Effects of G-MDSC exo on disease scores of paw swelling in CIA mouse; C:Effects of G-MDSC exo on disease progression of CIA mouse.

FIG. 7: Arginase (Arg)-1 carried by G-MDSC exo plays an important roleon alleviating autoimmune diseases. A: Detection result of Arg-1activity in G-MDSC exo and the suppression on Arg-1 activity by Arg-1inhibitor nor-NOHA. B: Effect of G-MDSC exo on the disease progressionof IBD mouse after inhibiting the activity of Arg-1; C: Effect of G-MDSCexo on the colon tissues of IBD mouse after inhibiting the activity ofArg-1; D: Effect of G-MDSC exo on the colonic inflammation afterinhibiting the activity of Arg-1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The technical solutions of this invention are described in conjunctionwith specific embodiments as follows but the invention is not limited tothese embodiments.

Example 1: G-MDSCs Sorting and the Preparation of Culture Supernatant

(1) The model of tumor-bearing mouse was established with the Lewis lungadenocarcinoma cell line (LLC): Lewis lung adenocarcinoma cells werecultivated in an incubator at 37° C. and 5% CO₂ in the medium (DMED withpH 7.2 and 10% fetal bovine serum). When the cell density is about 85%of the petri dish bottom area, cells are digested with 0.25% trypsin.Male 6-8 w C57BL/6 mouse were subcutaneously injected at the right sideof the abdomen in a dose of 3.0×10⁶ cells per mouse in logarithmicgrowth phase. The growth of tumors was observed after tumor planting.

(2) Establishment of CIA model: An equal volume of bovine collagen typeII (C II) and complete Freund's adjuvant are mixed at a ratio of 1:1 andgrind until the mixture is completely emulsified, The degree is thatdropping the emulsion into water and it is not loose (operation inice-bath). Emulsified C II (0.1 mL/mouse) was injected intradermally inthe base of the tail and make the emulsion to be absorbed completely.Using the emulsion of C II and incomplete Freund's adjuvant strengthenthe immune at day 21 after immunization, the day to immune is designatedas day 0.

(3) Isolation of splenocytes from tumor-bearing mouse or CIA mouse:After the model is constructed, mouse were sacrificed by eyebloodletting, then the spleen was sterilely removed and grinded in 0.22μm sieve. The suspension was filtered and centrifuged at 4° C., 500 gfor 5 min, the supernatant was discarded, and 5 mL of ACK lysis bufferis added into the cell pellet and mix well to lyse erythrocytes therein,hold for 5 min, and then add RPMI-1640 culture solution to 10 mL.Lastly, the cell suspension was centrifuged at 4° C., 500 g for 5 min,and the number of cells was calculated.

(4) G-MDSCs were isolated by magnetic bead: Splenocytes were resuspendedin 350 μL of PBE buffer per 10⁸ total cells, then 50 μL of FcR blockingreagent was added, mixing well and incubate for 10 minutes on the ice;adding anti-Ly-6G-Biotin (40 μL/10⁸ splenocytes), mixing well andincubate for 30 minutes on the ice, mixing every 10 minutes; adding 10mL of PBE buffer and centrifuge at 500 g for 5 minutes at 4° C.,discarding the supernatant; adding anti-Ly-6G-Biotin beads (50 μL/10⁸splenocytes), mixing well and incubate for 30 minutes on the ice, mixingevery 10 minutes; adding 10 mL of PBE buffer and centrifuge at 500 g for5 minutes at 4° C., discarding the supernatant; adding 500 μL of PBEbuffer and mixing well; placing MACS sorting column on VarioMACSseparator, rinsing the sorting column with 3 mL of PBE buffer; addingcell suspension onto the sorting column, washing column with 9 mL of PBEbuffer for 3 times after the first drop of the suspension outflows,removing the column from the separator and adding 5 mL of PBE onto thecolumn, pushing column bolt, squeezing out the cells bonding to thecolumn, collecting the cell suspension. Then, G-MDSCs were acquired.

(5) Purity analysis of G-MDSCs: 1×10⁶ G-MDSCs were collected in EP tubesand resuspended with 1 mL of PBS, and the cell suspension wascentrifuged at 4° C., 500 g for 5 min. The supernatant was discarded,100 μL PBS were left and resuspended, adding 0.5 μL of anti-Ly-6Gantibody and 0.5 μL anti-CD11b antibody and incubating at 4° C. for 30min, and then resuspending with 1 mL of PBS, centrifuging at 4° C., 500g for 5 min, discarding the supernatant and then adding 200 μL of PBS toresuspend. The expression of cell surface molecules detected by FACS,and the results was showed in FIG. 1. G-MDSCs are CD11b and Ly-6Gdouble-positive cells. The present invention uses immune magnetic beadsto sort G-MDSCs from tumor-bearing mouse spleen. The purity of G-MDSCwas detected by FACS, and the purity of MDSCs was >95%.

(6) Preparation of the culture supernatant of G-MDSCs: The sortedG-MDSCs were resuspended in RPMI-1640 culture solution containing 10% offetal bovine serum (that had been ultra-centrifuged at 100,000 g for 16h), inoculating onto 24-well plate at 1.5×10⁶ per well, the total volumeis 1 ml per well, incubating at 37° C. and 5% CO₂ for 24 h. The culturesupernatant of G-MDSC was harvested by centrifuging at 4° C., 300 g for20 min.

Example 2: Preparation of G-MDSC Exo and Detecting of ProteinConcentration

(1) The harvested G-MDSCs supernatant was centrifuged at 4° C., 1000 gfor 30 min, the supernatant was collected and centrifuged at 4° C.,10000 g for 30 min. The supernatant was transferred to anultrafiltration centrifugal tube with MWCO 100 kDa and was centrifugedat 1500 g for 30 min, and the concentrated liquid in the tube wascollected.

(2) G-MDSC exo was extracted by ExoQuick-TC™ Exosome Kit purchased fromSBI as follows: The concentrated liquid collected in step (1) was mixedwith ExoQuick-TC™ Exosome reagent (v/v=5:1), the mixture was vibratedand followed with a standing at 4° C. for more than 12 h and centrifugedat 4° C., 1000 g for 30 min, the precipitate was G-MDSC exo. G-MDSC exowas dissolved in PBS, dispensed to EP tube, and stored at −80° C. forsubsequent testing.

(3) Determine the protein concentration of G-MDSC exo by using the BCAProtein Assay Kit: The G-MDSC exo suspension was mixed with the lysisbuffer (RIPA:PMSF=250:1) at equivalent volume and incubated for 1 h onice, shake it every 10 min Finally, the mixture was centrifuged at 4°C., 12000 g for 15 min, and the supernatant was collected. The proteinconcentration in the lytic exosomes supernatant was detected accordingto the manufacture's instructions.

Example 3: Identification of G-MDSC Exo

(1) Observing the morphology of G-MDSC exo through transmission electronmicroscopic: 20 μL of G-MDSC exo suspension were dropped on a 3 mmdiameter of sample loading copper mesh, and rest for 2 minutes at roomtemperature; using filter paper to sip up the liquid gently, and drop 2%of phosphotungstic acid at pH 6.8 on the copper mesh and negativelystaining for 1 min, using filter paper sip up the dye liquid and driedunder incandescent light bulb. G-MDSC exo were observed as circular orelliptic micro-capsule structure having envelope by transmissionelectron microscopy, and the intracavity has low electron densitycomponents with particle size of 30-150 nm. The result is showed inFIGS. 2A and 2B. FIG. 2A shows the morphology of G-MDSC exo, the G-MDSCexo were observed as circular or elliptic micro-capsule structure havingcomplete envelope by transmission electron microscopy, and theintracavity has low electron density components. FIG. 2B shows thefrequency of particle-size distribution of G-MDSC exo. The particle sizeof G-MDSC exo observed by transmission electron microscope isdistributed at the range of 30-150 nm.

(2) The detection of CD63 molecules contained in exosomes and proteinsCalnexin contained in mitochondria by Western blot: preparing 5% ofspacer gel and 12% of separation gel, the denatured G-MDSC exo wasloaded at 250 μg. After 100V constant voltage electrophoresis, and 350mA constant current for 90 min, 5% defatted milk encloses the PVDF filmfor 1 h, and incubated with CD63 monoclonal antibody or calnexinmonoclonal antibody overnight at 4° C. The PVDF membrane was washed withTBS/T for 10 min and repeated 3 times. Then the PVDF membrane wasincubated with Horse Radish Peroxidase (HRP) conjugated anti-mousesecond antibody for 30 min at 37° C. The PVDF membrane was washed withTBS/T for 10 min, repeated 3 times and exposed and developed withImageQuant LAS 4000 gel imaging system, the result is as shown in FIG.2C. In FIG. 2C, the western blot shows that G-MDSC exo express CD63molecules and do not express mitochondria related calnexin molecules.

Example 4: G-MDSC Exo Suppress T Cell Proliferation and DTH Response

(1) The effect of G-MDSC exo on CD4⁺ T cell proliferation was detectedwith 3H-TdR incorporation method: CD4⁺ T cells were isolated. 6-8 weekmale C57BL/6 mouse were sacrificed by breaking the neck. The spleen wasremoved and ground sterile, and splencytes suspension was prepared. Thesupernatant was discarded after being centrifuged at 4° C., 500 g, for 5min. The cell sediment was added into 5 ml of ACK and stood for 5 min.Then 5 ml RPMI-1640 was added into suspension and centrifuging at 4° C.,500 g for 5 min. The precipitate was dissolved with 10 ml of PBEfollowed by centrifuging at 4° C., 500 g for 5 min and discarding thesupernatant. 15 μl anti-CD4-MicroBeads was added into cells suspensionat 1.0×10⁷ CD⁴⁺ T cell. The cells suspension was placed on the ice for30 min and mixed every 10 minutes. 10 ml of PBE was added to wash cellsand followed by centrifuging at 4° C., 500 g for 5 min and discardingthe supernatant. 500 μl of PBE was added into the precipitation and cellsuspension was prepared. Cell sorting column was put on the VarioMACSseparator and rinsed with 3 ml of PBE. The cells suspension was addedinto cell sorting column and 9 ml of PBE was used to wash sorting columnafter the first drop of suspension outflows. The column is removed and 5ml of PBE was added. Push the stud and collect the cell suspensionflowed out from the sorting column CD4⁺ T cells are obtained. CD4⁺ Tcells in 200 μl cell culture medium were inoculated into 96-well plateat 5×10⁵CD4⁺ T cells per well. Under the presence of anti-CD3 mAb andanti-CD28 mAb, different doses of G-MDSC exo were added into the wells.Cells were cultured with RPMI-1640 culture solution at pH 7.2 whichcontains 10% fetal bovine serum (after 100000 g×16 h centrifugal) under5% CO₂ atmosphere at 37° C. for 72 h, and [³H]-thymidine (1 μCi/well)was added. After 16 h. the counts per minute (CPM) values of variouswells were detected with an LS6500 Multi-Purpose Scintillation Counter.

(2) Observing inhibition effects of G-MDSC exo on CD4⁺ T cellsproliferation by mouse DTH model: DTH model was induced in 6-8 w maleC57BL/6 mouse, the mice were divided into normal control group (NC), DTHgroup, Neu exo treatment group, G-MDSX exo treatment group, and 6 mousesper group. In brief, C57BL/6 mouse were first immunized by intradermalinjections of 200 μl of CFA-emulsified OVA peptide at a finalconcentration of 1 mg/ml at the tail root and back. Seven days afterimmunization, each mouse was stimulated by right footpad injection of 30μl of OVA peptide at a concentration of 20 mg/ml. Footpad thickness wasmeasured at 24 h, 48 and 72 h after stimulating. The degree of footpadswelling in each group was calculated according to the judgment standardof DTH reaction. The result shows that degree of footpad swelling inG-MDSC exo treatment group is milder than the DTH group and Neu exotreatment group (FIG. 3B), and is statistically significant (p<0.05).These results implied that G-MDSC exo could suppress CD4⁺ T cells invivo.

The level of the DTH response was determined as follows (take 24 h forexample):

Swelling degree of footpad=(footpad thickness after OVA injection for 24h [min]−footpad thickness before OVA injection [mm])−(footpad thicknessafter PBS injection for 24 h [min]−footpad thickness before PBSinjection [mm]).

FIG. 3 shows the result of G-MDSC exo inhibiting CD4⁺ T cells, wherein,FIG. 3A shows the effect of G-MDSC exo on CD4⁺ T cell proliferationobserved by 3H-TdR incorporation method. The result shows that G-MDSCexo suppress CD4⁺ T cell proliferation in dose-dependent manner. FIG. 3Bshows the effect of G-MDSC exo on delayed type hypersensitivity observedby the DTH model. Swelling degree of footpad was measured afterOVA-stimulating 24 h, 36 h, and 72 h. The results show that the footpadthickness in G-MDSC exo treatment group is lower than that in othergroups. The results show that G-MDSC exo can suppress DTH reaction invivo.

Example 5: G-MDSC Exo Promote Cell Proliferation from CD4⁺ T Cells toTreg Induced by TGF-β in Dose-Dependent Manner

The magnetic beads sorting of CD4⁺ T cells was same the as example 4.2×10⁶/ml CD4⁺ T cells in 1 ml cell culture medium were inoculated in24-well plate and TGF-β inducing polarization of CD4⁺ T cells to Treg,adding different dose of G-MDSC exo under the presence of anti-CD3 mAband anti-CD28 mAb. CD25⁺Foxp3⁺ T cells were analyzed by FACS afterculturing for 3 days. The result shows that, G-MDSC can promote cellproliferation from CD4⁺ T cells to Treg induced by TGF-β (FIG. 4A) indose-dependent manner (FIG. 4B).

FIG. 4 shows the result of G-MDSC exo promote cell proliferation fromCD4⁺ T cells to Treg induced by TGF-β in dose-dependent manner. FIG. 4Ashows G-MDSC exo promote cell proliferation from CD4⁺ T cells to Treginduced by TGF-β at different concentration The results show that G-MDSCexo can promote Treg proliferation from CD4⁺ T cells under differentdoses of TGF-β. FIG. 4B shows G-MDSC exo promote cell proliferation fromCD4+ T cells to Treg cells induced by TGF-β in dose-dependent manner.These results show that Treg proliferation induced by TGF-β is increasedas the concentrations of G-MDSC exo increased.

Example 6: Treatment Efficacy of G-MDSC Exo on IBD Mouse

(1) Preparation of 2.5% DSS and induction of inflammatory bowel disease:25 g of Dextran Sulfate Sodium (DSS) was dissolved in 1 L of doubledistilled H₂O and cooled after autoclaving 6-8 w male C57BL/6 mousecontinuously drink 2.5% DSS solution for 9 days in free way to induceIBD.

(2) Mice are divided into four groups (6 mice per group) as follows:

Normal control group (NC): Mouse drinks double distilled water freely.

IBD group: Mouse drink 2.5% DSS solution without any other treatment.

Neu exo treatment group: Mice were treated with Neu exo throughintraperitoneal injection (30 μg/mouse) on days 2, 4, and 6 after DSSdrinking.

G-MDSC exo treatment group: Mice were treated with G-MDSC exo throughintraperitoneal injection (30 μg/mouse) on days 2, 4, and 6 after DSSdrinking.

Note: The day when drinking DSS is counted as day 0.

(3) Assessing IBD progression: the weight, stool property andhematochezia were monitored daily and graded DAI. Scoring criteria areas follows:

Weight change: <1% is 0 point, 1-5% is 1 point, 5-10% is 2 points,10-15% is 3 points, >15% is 4 points;

Stool: normal is 0 point, loose stool is 2 points, shapeless diarrhea is4 points;

Hematochezia: no is 0 point, visible blood is 4 points;

The sum of the points in each group divided by 3 is the final score.

(4) Observe colon specimens: Mouse were sacrificed on day 9 afterinducing IBD disease by eye bloodletting, the abdominal cavity of mousewas opened, colon tissue between the end of anus rectum and distal endof caecum was isolated, and then the extent of swelling and the lengthof colon tissues were observed.

(5) Pathology analysis of colon tissue of mouse in all groups: Colontissue between the end of anus rectum and distal end of caecum isisolated, the colons were fixed in 10% formalin solution,paraffin-embedded and stained with hematoxylin and eosin (H&E), and thepathological damage and degree of inflammation was observed under amicroscope.

(6) Experimental results: Evaluate the disease of the colon of mouse indifferent groups through scoring of weight loss, stool property andhematochezia, the damage of the colon of mouses in all groups isobserved by observing the outward appearance of colon, colonhistopathological staining. The results showed that the clinical diseasescore of G-MDSC exo-treatment mouse is significantly lower (FIG. 5A), aswell as decreased swelling and shorten of colon (FIG. 5B), and decreasedhistological damage, inflammatory cell infiltration and range ofinflammatory damage (FIG. 5C).

FIG. 5 shows the protective role of G-MDSC exo in IBD mouse. The effectof G-MDSC exo on IBD disease progression is shown in FIG. 5A. Thedisease progression was scored by body weight, stool property andhematochezia situation. The result shows that the clinical score ofmouse in G-MDSC exo-treatment group was lower than IBD group or Neuexo-treatment group. Moreover, this difference is statisticallysignificant (P<0.05) (FIG. 5A). The effect of G-MDSC exo on the colontissues of IBD mouse is shown in FIG. 5B. The degree of swelling andshorten of colon in G-MDSC exo-treatment group were lower than that ofIBD group or Neu exo-treatment group (FIG. 5B). The effect of G-MDSC exoon degree of inflammation of colon tissue is shown in FIG. 5C. Theinflammatory cell damage on colon tissue in G-MDSC exo-treatment groupare lower than that in IBD mouse or Neu exo-treatment group throughobserving the pathological staining of colon tissue of mice in allgroups (FIG. 5C). These results show that G-MDSC exo have protection forIBD mice.

Example 7: The Therapeutic Effect of G-MDSC Exo on CIA Mouse

The therapeutic effect of G-MDSC exo on CIA mouse: Mice were treatedintravenously on tail with G-MDSC exo on the 18 days and 24 days afterfirst immunization, the flowchart is shown in FIG. 6A. We found that theseverity of arthritis was significantly reduced in mouse treated withG-MDSC exo (FIG. 6B), and the time of onset is significantly delayed(FIG. 6C). As shown in FIG. 6B, the swelling could be observed on days27 after the initial immunization in untreated group, and the same casein G-MDSC exo treated group was observed on days 33. In addition, ondays 42 after immunization, the joint score was up to 10 in untreatedgroup, while the score of G-MDSC exo treated group was only about 4.

Example 8: G-MDSC Exo Carrying Arg-1 Play an Important Role inAttenuating Autoimmune Diseases

(1) The activity detection of Arg-1 in G-MDSC exo and inhibition effectof nor-NOHA on the Arg-1 activity in G-MDSC exo: The sorted G-MDSCs aresame to example 1. Inoculate 2×10⁶ G-MDSCs per well in 24 well plate,and add 1 ml of RPMI-1640 medium and culture at 37° C., 5% CO₂ for 16 h.The culture supernatant is collected and used for preparing G-MDSC exo,then the G-MDSC exo was lysed (the same with example 2), and the G-MDSCexo activity was measured with the arginase assay kit from eBiosciencecompany. 5 mg of nor-NOHA powder was dissolved with 1 ml of DMSO, 5mg/ml nor-NOHA solution is prepared. Experimental groups were treated asfollows:

G-MDSC group: G-MDSCs were cultured, and cells are collected.

(G-MDSC+DMSO) group: G-MDSC is cultured after adding 7W of DMSO, andcells were collected.

(G-MDSC+NN) group: G-MDSC is cultured after adding 7W of nor-NOHA, andcells were collected.

G-MDSC exo group: G-MDSC is cultured, culture supernatant was collected.

(G-MDSC+DMSO) exo group: G-MDSC is cultured after adding 7W of DMSO,culture supernatant was collected.

(G-MDSC+NN) exo group: G-MDSC is cultured after adding 7W of nor-NOHA,culture supernatant was collected.

The result shows that Arg-1 is contained in G-MDSC exo (FIG. 7A), andArg-1 activity could be inhibited by NN.

(2) G-MDSC exo plays a protective role in autoimmune disease by Arg-1therein. The present example observes that Arg-1 plays an important rolein protecting IBD mediated by G-MDSC exo through IBD model. According tothe requirement of experiment, the IBD mice are divided into Neu exotreatment group, G-MDSC exo treatment group, (G-MDSC+NN) exo treatmentgroup, and (G-MDSC+DMSO) exo treatment group. The method of configuringthe model, treating method and observing index are the same with example5.

The results show that Arg-1 involves in the protective role of G-MDSCexo in autoimmune disease. FIG. 7B shows that the effects of (G-MDSC+NN)exo treatment group on disease progression of IBD mouse, clinic score of(G-MDSC+NN) exo treatment group is obviously higher than that of G-MDSCexo treatment group by scoring of weight change, stool property andhematochezia. The difference was statistically significant. FIG. 7Cshows the effect of (G-MDSC+NN) exo on the colon tissues of IBD mouse.The result shows that the degree of swelling and shortening process in(G-MDSC+NN) exo-treatment group are obviously higher than that in G-MDSCexo-treatment group. FIG. 7D shows the effect of (G-MDSC+NN) exo on theinflammation of colon tissues of IBD mouse. The degree of inflammatorycell damage on colon tissue and inflammatory infiltration in (G-MDSC+NN)exo-treatment group are more severe than that in G-MDSC exo-treatmentgroup. These results strongly supported that Arg-1 played an importantrole in G-MDSC exo mediated protective roles in autoimmune diseases.

The invention claimed is:
 1. A method of-treating a patient with anautoimmune disease comprising isolating G-MDSCs from cancer individualsor autoimmune disease individuals to obtain an isolated G-MDSC cellpopulation having a purity of greater than 95%, culturing said G-MDSCcell population and isolating exosomes from the culture supernatant toprovide G-MDSC derived exosomes, and administering to the patient aneffective amount of said G-MDSC derived exosomes, wherein the exosomesare membrane vesicles containing the lipid component of cell membrane ofderived sources, the exosomes also have nonspecific components ofG-MDSCs and specific active components including protein and nucleicacid in G-MDSCs, and are composite informosomes of subcellular structurefor intercellular information transmission, and wherein the exosomescontain Arg-1 and exhibit immunosuppressive properties.
 2. The methodaccording to claim 1, wherein, the autoimmune disease is inflammatorybowel disease.
 3. The method according to claim 1, wherein, theautoimmune disease is autoimmune arthritis.
 4. A method of treating apatient having an autoimmune disease, the method comprising: isolatingG-MDSCs from cancer individuals or autoimmune disease individuals toobtain an isolated G-MDSC cell population having a purity of greaterthan 95%, culturing said G-MDSC cell population and isolating exosomesfrom the culture supernatant to provide G-MDSC derived exosomes, andadministering to the patient an effective amount of said G-MDSC derivedexosomes, wherein the exosomes have circular or elliptic micro-capsulestructure with complete envelope, and an intracavity having low electrondensity components, the exosomes also having a diameter of 40-100 nm andcarrying biologically active ingredients, wherein the exosomes aremembrane vesicles containing the lipid component of cell membrane ofderived sources, the exosomes also have nonspecific components ofG-MDSCs and specific active components including protein and nucleicacid in G-MDSCs, and are composite informosomes of subcellular structurefor intercellular information transmission, and wherein the exosomescontain Arg-1 and exhibit immunosuppressive properties.
 5. The methodaccording to claim 4, wherein, the autoimmune disease is inflammatorybowel disease.
 6. The method according to claim 4, wherein, theautoimmune disease is autoimmune arthritis.
 7. A method of treating apatient having a disease caused by delayed type hypersensitivityreaction comprising: isolating G-MDSCs from cancer individuals orautoimmune disease individuals to obtain an isolated G-MDSC cellpopulation having a purity of greater than 95%, culturing said G-MDSCcell population and isolating exosomes from the culture supernatant toprovide G-MDSC derived exosomes, and administering to the patient aneffective amount of said G-MDSC derived exosomes, wherein the exosomesare membrane vesicles containing the lipid component of cell membrane ofderived sources, the exosomes also have nonspecific components ofG-MDSCs and specific active components including protein and nucleicacid in G-MDSCs, and are composite informosomes of subcellular structurefor intercellular information transmission, and wherein the exosomescontain Arg-1 and exhibit immunosuppressive properties.